This was the fourth year for this project, which is using biochemical and biophysical methods to examine conformational changes related to transport in glutamate transporters. These proteins are of critical importance in the central nervous system, where they play important roles in clearing neurotransmitters from synapses and in shaping the electrical activity of post-synaptic neurons. These transporters have been implicated as playing roles in a variety of diseases, including ALS, Alzheimers disease, and exitotoxicity. It is critical to understand the fundamental mechanisms by which there transporters function because such knowledge could lead to the development of therapeutic agents active against these proteins. We seek to analyze the dynamic movements of the functioning transporter on the way to a detailed understanding of its mechanism. Our approach is to analyze the details of transport in model glutamate transporters obtained from bacteria. These can be expressed and purified in large quantities and are amenable to biophysical methods not available for their mammalian cousins. We previously discovered that the bacterial glutamate transporters display a chloride transport activity which is stoichiometrically uncoupled from glutamate uptake. This chloride transport activity is similar to one which is important in the mammalian transporters and suggests that the bacterial homologs provide an excellent structural model in which to study the process of chloride transport in these proteins. This year we completed our characterization of this chloride conductance demonstrating that it operates using similar parts of the protein as the eukaryotic glutamate transporters. We also found that, in contrast to the EAATs, no other ions besides Na+, Cl-, and the substrate are involved in the transport cycle. Our studies on conformational changes in these proteins continued this year;using limited proteolysis and cysteine scanning mutagenesis, we found evidence of a significant substrate-induced conformational change and localized the change to a novel protein heretofore not expected to participate in the transport process. We further found that the protein movement is an essential part of the transport process. Also, we are using EPR spectroscopy to probe transport-related conformational changes and have found substantial evidence for major movements of the protein, which oppose and separate pairs of spin labels with changes in substrate and sodium. We are currently exploring the extent of these changes and their kinetics.